Methods, systems and solid compositions for reagent delivery

ABSTRACT

Methods, systems, and compositions featuring a solid, dissolvable reagent composition for delivering the reagent, such as an antibody, probe, chromogen, etc., to a sample. The present invention also features methods of producing said compositions, and automated systems featuring the use of the solid, dissolvable reagent compositions. The solid, dissolvable reagent composition may comprise a water-soluble polymer film, such as a polyvinyl alcohol film, infused with the reagent, wherein when applied to the sample, the water-soluble polymer film with reagent contacts the sample (e.g., tissue) and dissolves.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of International PatentApplication No. PCT/US2017/067835 filed Dec. 21, 2017, which claimspriority to and the benefit of U.S. Provisional Application No.62/437,545, filed Dec. 21, 2016. Each of the above patent applicationsis incorporated herein by reference as if set forth in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to antibodies and reagent delivery, moreparticularly to methods, systems, and compositions for antibody deliveryusing solid compositions such as a solid, dissolvable single-doseantibody composition.

BACKGROUND OF THE INVENTION

Current reagent dispenser systems for automated cell stainers aregenerally produced to accommodate at least 50 tests, and the reagentsgenerally have a limited shelf life (e.g., 9-24 months). Many reagentsmust be kept at 4° C. The dispensers are designed to deliver precisedrops (e.g., 100 μL) of solutions (comprising antibodies, probes and/orother reagents) to target slides; however, mis-dispense and residueformation can potentially lead to inaccurate volumes of liquid beingdelivered and subsequent slide staining failures.

SUMMARY OF INVENTION

It was surprisingly discovered reagents, such as antibodies, could beincorporated into a water-soluble polymer (such as polyvinyl alcohol(PVA) such that the reagents were not degraded or destroyed. Forexample, as described below, antibodies were safely heated with PVAwithout causing damage to the functionality of the antibody, creating asolid, dissolvable reagent composition.

The present invention features compositions comprising a solid,dissolvable reagent composition for delivering said reagent (e.g.,antibodies, probes, buffers, chromogens, counterstains, enzymes, nucleicacids, etc.) to a sample. The compositions comprise a water-solublepolymer (e.g., a film, a pouch, a wafer, etc.) and the reagent. In someembodiments, the reagent is infused, printed, embedded, or encapsulatedin the water-soluble polymer. When the composition is applied to asample, the water-soluble polymer contacts the sample and dissolves,allowing the reagent to contact the sample. A non-limiting example of awater-soluble polymer is polyvinyl alcohol (PVA).

In some embodiments, the reagent (e.g., antibody, probe, stain, enzyme,etc.) is infused between at least two layers of the water-solublepolymer film. In some embodiments, the reagent is inkjet-printed ontothe surface of the water-soluble polymer film. In some embodiments, thereagent is embedded into the water-soluble polymer film.

The present invention also features methods of producing saidcompositions and methods of using said compositions (applications of thecompositions), e.g., histochemical methods, methods of introducingantibodies to a sample, etc. The present invention also features systems(e.g., automated systems, manual systems) featuring the use of saidcompositions. For example, the present invention features methods,systems, and compositions for delivering antibodies, stains, or otherreagents to a tissue sample adhered to a slide. The solid, dissolvablereagents of the present invention may be used in an automated stainingmachine. The present invention is not limited to automated methods andsystems. In some embodiments, the compositions of the present inventionare applied to a sample in a manual manner.

The sample may be of any type appropriate for the application. Forexample, in some embodiments, a tissue sample is an appropriate samplefor histological examination on a slide. In one embodiment, the tissuesample may be immobilized on a slide and further processed with anautomated slide stainer, for instance an automated IHC/ISH slidestainer. In some embodiments, the tissue sample may be taken from abiopsy or a surgical resection. The tissue sample may further comprise,for example, a section of a cancerous or healthy tissue. In addition,the tissue sample may be taken from a liquid cytology sample. Note thesample is not limited to a tissue sample.

The present invention is not limited to the specific steps, components,compositions, etc. For example, other water-soluble substrates may beused. Various different temperatures, humidity, or other conditions maybe considered. Other reagents may be used, etc.

Without wishing to limit the present invention to any theory ormechanism, it is believed that the solid, dissolvable compositions (andmethods and systems herein) are advantageous. For example, thecomposition may be kept at room temperature (even left on instrumentsfor long periods of time) and that the composition may be more stablethan the reagent in a liquid form. Other advantages may include theelimination of the need to rehydrate the reagent prior to application onthe sample (bypass of a hydration step). The number of steps needed toperform certain experiments can be reduced. In the case of an automatedsystem, the number of dispensers required is reduced. Further, one couldperform small numbers of experiments (e.g., 1 slide, 5 slides) or largenumbers (e.g., 50 slides).

Note the present invention also features compositions made from thewater-soluble polymer (e.g., PVA) and reaction buffer (or other buffer)as an alternative to the water-soluble polymer (e.g., PVA) andwater/ethanol.

The present invention provides an automated staining system comprising:a base for supporting a sample (e.g., a tissue section, a tissuesample); a liquid dispenser for aliquoting a liquid onto the sample onthe base; and a solid composition for depositing onto the sample on thebase. The solid composition dissolves onto the sample and therebydepositing the reagent on the sample. In some embodiments, when thesolid composition contacts the sample, the composition dissolves therebydepositing the reagent on the sample. The solid composition may bedeposited manually. In some embodiments, the solid composition isdeposited in an automated manner. For example, in some embodiments, thesystem comprises a solid reagent dispenser for depositing the solidcomposition onto the sample. In some embodiments, the system comprises aholding container for storing or holding the solid composition. In someembodiments, the solid reagent dispenser retrieves a solid reagent fromthe holding container and moves it to the sample on the base.

In certain embodiments, the solid composition comprises a water-solublepolymer and a reagent embedded therein, the reagent is selected from agroup consisting of: an antibody, an antibody fragment, a protein, anucleic acid, a chromogen, a stain, a counterstain, a lipid, acarbohydrate, an enzyme, a buffer, or a combination thereof; the solidcomposition is deposited on the sample manually.

In some embodiments, the liquid dispenser aliquots the liquid onto thesample on the base before the solid composition is deposited onto thesample on the base. In some embodiments, the liquid dispenser aliquotsthe liquid onto the sample on the base after the solid composition isdeposited onto the sample on the base.

The system may be capable of holding and processing multiple slides. Forexample, in some embodiments, the system holds and processes at least 20samples. In some embodiments, the system holds and processes at least 50samples. The automated staining system may for introducing a stain tothe sample. In some embodiments, the system may be for performing ahistochemistry assay on the sample. In some embodiments, the system maybe for performing in situ hybridization on the sample. In someembodiments, the system may be for automated DNA amplification orsequencing.

In certain embodiments, the system can deliver solid compositions orboth solid and liquid compositions to the sample.

In certain embodiments, the system further comprises an imageacquisition system, an image analysis system, or both an imageacquisition system and image analysis system.

In some embodiments, the water-soluble polymer comprises polyvinylalcohol (PVA), dextran, hydroxypropyl cellulose, poly (acrylic acidsodium salt), poly (ethylene glycol), poly (methylacrylic acid sodiumsalt), poly(styrenesulfonic acid sodium salt), pullulan, or acombination thereof. In some embodiments, the composition comprises twoor more reagents. In some embodiments, the water-soluble polymer is afilm. In some embodiments, the reagent is embedded or infused in thewater-soluble polymer. In some embodiments, the reagent is printed onthe water-soluble polymer. In some embodiments, the reagent isencapsulated by the water-soluble polymer. In some embodiments, thereagent is encapsulated by two or more layers of the water-solublepolymer. In some embodiments, the solid composition further comprisessalts from a buffer. In some embodiments, the solid composition furthercomprises a buffering agent. In some embodiments, the sample comprises atissue sample, e.g., a tissue section. In some embodiments, the sampleis mounted on a slide.

The present invention also provides a method of depositing a reagentonto a sample. In some embodiments, the method comprises: in a systemcomprising a base for supporting a sample and a liquid dispenser foraliquoting a liquid onto the sample on the base: placing a sample ontothe base of the system; and depositing a solid composition onto thesample on the base, wherein when the solid composition contacts thesample, the composition dissolves thereby depositing the reagent on thesample. In some embodiments, the solid composition comprises awater-soluble polymer a reagent embedded therein, the reagent isselected from a group consisting of: an antibody, an antibody fragment,a protein, a nucleic acid, a chromogen, a stain, a counterstain, alipid, a carbohydrate, an enzyme, a buffer, or a combination thereof.

In some embodiments, the solid composition is deposited on the samplemanually. In some embodiments, the solid composition is deposited on thesample in an automated manner. In some embodiments, the system furthercomprises a holding container for holding the solid composition. In someembodiments, the system comprises a solid reagent dispenser fordepositing the solid composition onto the sample. In some embodiments,the solid reagent dispenser retrieves a solid reagent from the holdingcontainer and moves it to the sample on the base.

In some embodiments, the liquid dispenser aliquots the liquid onto thesample on the base before the solid composition is deposited onto thesample on the base. In some embodiments, the liquid dispenser aliquotsthe liquid onto the sample on the base after the solid composition isdeposited onto the sample on the base.

In certain embodiments, the system is capable of holding and processingmultiple samples. For example, in some embodiments, the system can holdand process at least 20 samples. In some embodiments, the system canhold and process at least 50 samples.

In certain embodiments, the system is for introducing a stain to thesample. In some embodiments, the system is for performing ahistochemistry assay on the sample. In some embodiments, the system isfor performing in situ hybridization on the sample. In some embodiments,the system is for automated DNA amplification or sequencing.

In some embodiments, the system can deliver solid compositions or bothsolid and liquid compositions to the sample. In some embodiments, thesystem further comprises an image acquisition system, an image analysissystem, or both an image acquisition system and image analysis system.

In certain embodiments, the water-soluble polymer comprises polyvinylalcohol (PVA), dextran, hydroxypropyl cellulose, poly (acrylic acidsodium salt), poly (ethylene glycol), poly (methylacrylic acid sodiumsalt), poly(styrenesulfonic acid sodium salt), pullulan, or acombination thereof. In some embodiments, the composition comprises twoor more reagents. In some embodiments, the water-soluble polymer is afilm. In some embodiments, the reagent is embedded or infused in thewater-soluble polymer. In some embodiments, the reagent is printed onthe water-soluble polymer. In some embodiments, the reagent isencapsulated by the water-soluble polymer. In some embodiments, thereagent is encapsulated by two or more layers of the water-solublepolymer. In some embodiments, the solid composition further comprisessalts from a buffer. In some embodiments, the solid composition furthercomprises a buffering agent. In some embodiments, the sample comprises atissue sample, e.g., a tissue section. In some embodiments, the sampleis mounted on a slide.

The present invention provides an automated apparatus for delivering areagent to a sample. In certain embodiments, the system comprises anautomated machine for holding at least sample (e.g., a sample on aslide, e.g., a tissue sample mounted on a slide); a dispensing mechanismin the automated machine for placing a solid composition on the sample,wherein when the composition contacts the sample, the compositiondissolves thereby depositing the reagent on the sample. The solidcomposition comprises a water-soluble polymer and a reagent selectedfrom: an antibody, an antibody fragment, a protein, a nucleic acid, achromogen, a stain, a counterstain, a lipid, a carbohydrate, an enzyme,a buffer, or a combination thereof.

The apparatus may comprise a holding container (e.g., in the automatedmachine, connected to the automated machine) holding the solidcomposition. The solid composition may be stored in the automatedmachine until dispensed onto the sample. In some embodiments, the solidcomposition is stored in a holding container.

In certain embodiments, the automated machine is an automated systemsuch as those described herein for introducing a stain to a sample, forperforming histochemical assays, for performing in situ hybridization,etc. For example, specific examples of automated staining machines(e.g., IHC/ISH slide stainers) include: itelliPATH (Biocare Medical),WAVE (Celerus Diagnostics), DAKO OMNIS and DAKO AUTOSTAINER LINK 48(Agilent Technologies), BENCHMARK XT (Ventana Medical Systems, Inc.),BENCHMARK ULTRA (Ventana Medical Systems, Inc.), BENCHMARK GX (VentanaMedical Systems, Inc.), VENTANA H&E 600 (Ventana Medical Systems, Inc.),BENCHMARK Special Stains (Ventana Medical Systems, Inc.), VENTANADISCOVERY XT (Ventana Medical Systems, Inc.), VENTANA DISCOVERY ULTRA(Ventana Medical Systems, Inc.), Leica BOND, and Lab Vision Autostainer(Thermo Scientific). Automated staining machines (automated slidestainers) are also described by Prichard, Overview of AutomatedImmunohistochemistry, Arch Pathol Lab Med., Vol. 138, pp. 1578-1582(2014), incorporated herein by reference in its entirety. Additionally,Ventana Medical Systems, Inc. is the assignee of a number of UnitedStates patents disclosing systems and methods for performing automatedanalyses, including U.S. Pat. Nos. 5,650,327, 5,654,200, 6,296,809,6,352,861, 6,827,901 and 6,943,029, and U.S. Published PatentApplication Nos. 20030211630 and 20040052685, each of which isincorporated herein by reference in its entirety. The methods of thepresent invention may be adapted to be performed on any appropriateautomated staining machine (or automated slide processing machine).

In certain embodiments, the automated apparatus (e.g., automatedmachine) is for automated DNA or RNA amplification. In certainembodiments, the automated apparatus (e.g., automated machine) is forDNA sequencing.

The automated machine may be capable of holding and processing at least20 samples. The automated machine may be capable of holding andprocessing at least 50 samples.

The apparatus (e.g., automated machine) may be configured with one ormore dispensing mechanisms for delivering the solid composition to thesample, e.g., two or more dispensing mechanism, three or more, etc. Thedispensing mechanism(s) may be able to deliver more than one solidcomposition (e.g., two or more different solid compositions) to thesample. The apparatus (e.g., automated machine) may be configured todeliver solid compositions to the sample. The apparatus may beconfigured to deliver both solid and liquid compositions to the sample.

In some embodiments, the apparatus further comprises image acquisitionsystem, an image analysis system, or both an image acquisition systemand image analysis system. One example of a brightfield imager that cangenerate digitized tissue data is the iScan HT and DP200 (Griffin)brightfield scanner sold by Ventana Medical Systems, Inc., or anymicroscope having one or more objective lenses and a digital imager, aswell as a set of spectral filters. In some embodiments, the imagingapparatus is a digital pathology device as disclosed in InternationalPatent Application No.: PCT/US2010/002772 (Patent Publication No.:WO/2011/049608) entitled IMAGING SYSTEM AND TECHNIQUES or disclosed inU.S. Patent Application No. 61/533,114, filed on Sep. 9, 2011, entitledIMAGING SYSTEMS, CASSETTES, AND METHODS OF USING THE SAME. InternationalPatent Application No. PCT/US2010/002772 and U.S. Patent Application No.61/533,114 are incorporated by reference in their entirety. Otherexamples of commercially available slide scanners include: 3DHistechPANNORAMIC SCAN II; DigiPath PATHSCOPE; Hamamatsu NANOZOOMER RS, HT, andXR; Huron TISSUESCOPE 4000, 4000XT, and HS; Leica SCANSCOPE AT, AT2, CS,FL, and SCN400; Mikroscan D2; Olympus VS120-SL; Omnyx VL4, and VL120;PerkinElmer LAMINA; Philips ULTRA-FAST SCANNER; Sakura FinetekVISIONTEK; Unic PRECICE 500, and PRECICE 600x; VENTANA ISCAN COREO andISCAN HT; and Zeiss AXIO SCAN.Z1. The present invention is not limitedto the aforementioned image systems.

The present invention provides solid compositions comprising awater-soluble polymer and a reagent selected from: an antibody, anantibody fragment, a protein, a nucleic acid, a chromogen, a stain, acounterstain, a lipid, a carbohydrate, an enzyme, a buffer, or acombination thereof. In certain embodiments, the water-soluble polymercomprises polyvinyl alcohol (PVA), dextran, hydroxypropyl cellulose,poly (acrylic acid sodium salt), poly (ethylene glycol), poly(methylacrylic acid sodium salt), poly(styrenesulfonic acid sodiumsalt), pullulan, or a combination thereof. In certain embodiments, thecomposition comprises two or more reagents.

In some embodiments, the water-soluble polymer is a film. In someembodiments, the reagent is embedded or infused in the water-solublepolymer. In some embodiments, the reagent is printed on thewater-soluble polymer. In some embodiments, the reagent is encapsulatedby the water-soluble polymer. In some embodiments, the reagent isencapsulated by two or more layers of the water-soluble polymer. In someembodiments, the solid composition further comprises salts from abuffer. In some embodiments, the solid composition further comprises abuffering agent.

The present invention also provides methods for contacting a sample witha reagent, wherein the method comprises applying to the sample a solidcomposition according to the present invention, wherein the compositiondissolves upon contact with the sample thereby depositing the reagent onthe sample. The sample may be a tissue sample, e.g., a tissue samplemounted on a slide. The method may feature placing the sample in anautomated staining machine (e.g., as described above). The method mayfeature placing the sample in an automated apparatus (e.g., as describedabove).

The present invention also provides workflow methods for processing andpreparing a sample and subjecting the sample to an automated method suchas IHC or ISH on an automated staining machine using solid reagents (asdescribed herein). For example, the method may comprise preparing tissuesection from a tumor of a patient, e.g., sectioning a FFPE tissue sampleof a tumor of a patient using a microtome and mounting the tissuesection on a slide; histochemically staining the tissue section for atarget biomarker, wherein one or more reagents used to stain the tissuesection are solid reagents. The tissue section may be placed in anautomated staining machine, which automatically dispenses one or moresolid reagents onto the tissue section for the purpose of staining thetarget biomarker. The method may include staining the tissue section fora second target biomarker using one or more solid reagents in anautomated staining machine.

The present invention also provides automated staining systemscomprising at least one holding container for holding a solid reagentand an apparatus (e.g., a mechanical means, a dispensing arm) forobtaining a unit of the solid reagent from the holding container andplacing the solid reagent on a slide.

The present invention also features histochemical methods for labeling atarget biomarker in a tissue sample, wherein the methods comprisecontacting the sample with a solid composition of the present inventionto thereby introduce a reagent to the sample.

The present invention also features in situ hybridization methods forlabeling a target nucleic acid in a tissue sample, wherein the methodscomprise contacting the sample with a solid composition of the presentinvention to thereby introduce a reagent to the sample.

The present invention also features staining methods for staining atissue sample, wherein the methods comprise contacting the sample with asolid composition of the present invention to thereby introduce areagent to the sample.

The present invention also features nucleic acid (e.g., DNA, RNA)amplification methods for amplifying DNA, wherein the methods comprisecontacting the sample with a solid composition of the present inventionto thereby introduce a reagent to the sample for the purpose ofamplifying nucleic acid.

The present invention also features nucleic acid (e.g., DNA, RNA)sequencing methods, wherein the methods comprise contacting the samplewith a solid composition of the present invention to thereby introduce areagent to the sample for the purpose of sequencing nucleic acid.

The present invention also features kits comprising one or more solidcompositions. For example, a kit may comprise one or more of: a solidcomposition comprising a deparaffinization reagent, a solid compositioncomprising an antigen retrieval solution, a solid composition comprisinga primary antibody, a solid composition comprising a secondary antibody,a solid composition comprising a third antibody, a solid compositioncomprising a first detection reagent, a solid composition comprising asecond detection reagent, a solid composition comprising Hematoxylin,and a solid composition comprising a Bluing Reagent.

In certain embodiments, the kit may comprise reagents for histochemistry(e.g., immunohistochemistry). For example, in some embodiments, the kitmay comprise one or more of: a solid composition comprising adeparaffinization reagent, a solid composition comprising an antigenretrieval solution, a solid composition comprising a specific bindingagent for a target (e.g., an antibody), a solid composition comprising adetection reagent, a solid composition comprising a counterstain, asolid composition comprising a morphological stain, etc.

The kit may comprise reagents for staining, e.g., one or a combinationof: a solid composition comprising a counterstain, a solid compositioncomprising a morphological stain, a solid composition comprising achromogenic stain, etc.,

Examples of counterstains include chromogenic nuclear counterstains,such as hematoxylin (stains from blue to violet), Methylene blue (stainsblue), toluidine blue (stains nuclei deep blue and polysaccharides pinkto red), nuclear fast red (also called Kernechtrot dye, stains red), andmethyl green (stains green); non-nuclear chromogenic stains, such aseosin (stains pink); fluorescent nuclear stains, including 4′,6-diamino-2-pheylindole (DAPI, stains blue), propidium iodide (stainsred), Hoechst stain (stains blue), nuclear green DCS1 (stains green),nuclear yellow (Hoechst S769121, stains yellow under neutral pH andstains blue under acidic pH), DRAQ5 (stains red), DRAQ7 (stains red);fluorescent non-nuclear stains, such as fluorophore-labeled phalloidin,(stains filamentous actin, color depends on conjugated fluorophore).

Many morphological stains are known, including but not limited to,hematoxylin and eosin (H&E) stain and Lee's Stain (Methylene Blue andBasic Fuchsin).

In certain embodiments, the kit may comprise reagents for in situhybridization. For example, in some embodiments, the kit may compriseone or more of: a solid composition comprising a deparaffinizationreagent, a solid composition comprising an antigen retrieval solution, asolid composition comprising a nucleic acid probe, a solid compositioncomprising a detection reagent, etc.

In certain embodiments, the kit comprises one or more detectionreagents. Non-limiting examples of commercially available detectionreagents or kits comprising detection reagents include: VENTANAultraView detection systems (secondary antibodies conjugated to enzymes,including HRP and AP); VENTANA iVIEW detection systems (biotinylatedanti-species secondary antibodies and streptavidin-conjugated enzymes);OptiView detection systems (OptiView) (anti-species secondary antibodyconjugated to a hapten and an anti-hapten tertiary antibody conjugatedto an enzyme multimer); VENTANA Amplification kit (unconjugatedsecondary antibodies, which can be used with any of the foregoingVENTANA detection systems to amplify the number of enzymes deposited atthe site of primary antibody binding); OptiView Amplification system(Anti-species secondary antibody conjugated to a hapten, an anti-haptentertiary antibody conjugated to an enzyme multimer, and a tyramideconjugated to the same hapten; VENTANA DISCOVERY (e.g. DISCOVERY YellowKit, Discovery Purple Kit, Discovery Silver kit, DISCOVERY Red Kit,DISCOVERY Rhodamine Kit, etc.) VENTANA DISCOVERY OmniMap, VENTANADISCOVERY UltraMap anti-hapten antibody, secondary antibody, chromogen,fluorophore, and dye kits, each of which are available from VentanaMedical Systems, Inc. (Tucson, Ariz.); PowerVision and PowerVision+IHCDetection Systems (secondary antibodies directly polymerized with HRP orAP into compact polymers bearing a high ratio of enzymes to antibodies);and DAKO EnVision™+System (enzyme labeled polymer that is conjugated tosecondary antibodies).

In some embodiments, the automated apparatus (e.g., automated stainingmachine) can hold multiple solid compositions, e.g., solid compositionsfor each reagent involved in a particular assay, e.g., solidcompositions for each reagent involved in a particular staining method,histochemistry method, in situ hybridization method, DNA amplificationmethod, DNA sequencing method, etc.

Without wishing to limit the present invention to any theory ormechanism, it is believed that a discrete, single-dose deliverycomposition (and method) would have advantages, such as the potential tobe more precise and reliable, the potential to improve stability and/orshelf life of the reagent (e.g. antibody), the simplification of anautomated system (e.g., the instrument or dispenser would not berequired to accurately measure out and deliver small volumes of liquid),etc. Additionally, such compositions and methods may appeal tolow-throughput users such as those who do not have the run volume toutilize large amounts of rare or expensive reagents/assays before theexpiration date. Users could potentially increase their range of assaysif single-slide advanced staining kits and single-slide doses for rareand expensive reagents/assays were available. A solid delivery methodmay also reduce reagent waste. For example, in the case of an automatedsystem, it would not be necessary to prime the dispensers before eachrun. Single-serving compositions may also increase the ease of use(e.g., the reagents can be kept at room temperature, all requiredreagents may be included in one dispenser, no need to ensure that all ofthe required reagents needed for an assay have been loaded onto theinstrument, etc.) and reduce costs.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. Additional advantages and aspects ofthe present invention are apparent in the following detailed descriptionand claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The application or application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

FIG. 1A shows distribution of reagent-PVA solutions in trays in 500 μLdroplets for dehydration into a film in an oven at 37° C. for 1 hour.

FIG. 1B shows dissolving of a reagent wafer when applied to a buffer ona slide.

FIG. 2A is a photograph illustrating an antibody-PVA solutiondistributed in trays in 500 μL droplets for dehydration into a film.

FIG. 2B is a photograph of slides containing the antibody-PVA thin filmof FIG. 2A inserted into an automated slide stainer.

FIG. 3A is a photograph illustrating a PVA packet prototype filled withantibody diluted in glycerin.

FIG. 3B is a photograph illustrating the packet prototype of FIG. 3Aplaced on an automated slide stainer.

FIG. 4A is a photograph illustrating an inkjet-printed antibody on PVAfilm prototype mounted to the printing stage of the inkjet printer.

FIG. 4B is a photograph illustrating the inkjet printer used to printthe prototype of

FIG. 4A

FIG. 5A is a 1× micrograph (with 4× zoom inset) illustrating normalpancreas tissue stained with PTEN (SP218) antibody from 2% PVA antibodyembedded film applied during the Linker (secondary antibody) detectionstep.

FIG. 5B is a 1× micrograph (with 4× zoom inset) illustrating normalpancreas tissue stained with PTEN (SP218) antibody with a dispenser forthe Linker (secondary antibody) detection step as a control.

FIG. 5C is a 1× micrograph (with 4× zoom inset) illustrating normalpancreas tissue stained with PTEN (SP218) antibody from 2% PVA antibodyembedded film applied during the Multimer (tertiary antibody) detectionstep.

FIG. 5D is a 1× micrograph (with 4× zoom inset) illustrating normalpancreas tissue stained with PTEN (SP218) antibody with a dispenser forthe Multimer (tertiary antibody) detection step as a control.

FIG. 6A is a 1× micrograph (with 4× zoom inset) illustrating normalpancreas tissue stained with PTEN (SP218) antibody from 2% PVA DABembedded film applied during the DAB detection step.

FIG. 6B is a 1× micrograph (with 4× zoom inset) illustrating normalpancreas tissue stained with PTEN (SP218) antibody with a dispenser forthe DAB detection step as a control.

FIG. 6C is a 1× micrograph (with 4× zoom inset) illustrating normalpancreas tissue stained with PTEN (SP218) antibody from 2% PVA CopperReagent embedded film applied during the Copper Reagent detection step.

FIG. 6D is a 1× micrograph (with 4× zoom inset) illustrating normalpancreas tissue stained with PTEN (SP218) antibody with a dispenser forthe Copper Reagent detection step as a control.

FIG. 7A is a 1× micrograph (with 4× zoom inset) illustrating normalpancreas tissue stained with PTEN (SP218) antibody from 2% PVA BluingReagent embedded film applied during the Bluing Reagent counterstainstep.

FIG. 7B is a 1× micrograph (with 4× zoom inset) illustrating normalpancreas tissue stained with PTEN (SP218) antibody with a dispenser forthe Bluing Reagent counterstain step as a control.

FIG. 7C is a 1× micrograph (with 4× zoom inset) illustrating normalpancreas tissue stained with PTEN (SP218) antibody from 2% PVAHematoxylin II Reagent embedded film applied during the hematoxylincounterstain step.

FIG. 7D is a 1× micrograph (with 4× zoom inset) illustrating normalpancreas tissue stained with PTEN (SP218) antibody with a dispenser forthe hematoxylin counterstain step as a control.

FIG. 8A is a 1× micrograph (with 20× zoom inset) illustrating normalpancreas tissue stained with PTEN (SP218) antibody using a primaryantibody wafer, a linker wafer, a multimer wafer, a DAB wafer, a copperwafer, a bluing reagent wafer, and a hematoxylin wafer (made from 2% PVAin reaction buffer).

FIG. 8B is a 1× micrograph (with 20× zoom inset) illustrating normalpancreas tissue stained with PTEN (SP218) antibody with a dispenser foreach antibody and reagent steps as a control.

FIG. 9A is a micrograph illustrating normal prostate tissue stained withPTEN (SP218) antibody using 1% PVA antibody embedded film during thePTEN (SP218) primary antibody step.

FIG. 9B is a micrograph illustrating normal prostate tissue stained withPTEN (SP218) antibody using a dispenser as a control.

FIG. 9C is a micrograph illustrating 40× magnification of the image ofFIG. 9A.

FIG. 9D is a photograph illustrating 40× magnification of the image ofFIG. 9B.

FIG. 10A is a micrograph illustrating normal prostate tissue stainedwith PTEN (SP218) antibody using PVA packet filled with glycerin andantibody.

FIG. 10B is a micrograph illustrating normal prostate tissue stainedwith PTEN (SP218) antibody using a dispenser as a control.

FIG. 10C is a micrograph illustrating 40× magnification of the image ofFIG. 10A.

FIG. 10D is a micrograph illustrating 40× magnification of the image ofFIG. 10B.

FIG. 11A is a micrograph illustrating normal placenta tissue stainedwith CD34 (QBEnd/10) primary antibody printed onto PVA film.

FIG. 11B is a micrograph illustrating normal placenta tissue stainedwith CD34 (QBEnd/10) primary antibody delivered with a dispenser as acontrol.

FIG. 11C is a micrograph illustrating 40× magnification of the slide ofFIG. 11A.

FIG. 11D is a micrograph illustrating 40× magnification of the slide ofFIG. 11B.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical and/or bind the sameepitope, except for possible variant antibodies, e.g., containingnaturally occurring mutations or arising during production of amonoclonal antibody preparation, such variants generally being presentin minor amounts. The term “polyclonal antibody” refers to an antibodypreparation that typically includes different antibodies directedagainst different determinants (epitopes). In contrast to a polyclonalantibody, each monoclonal antibody of a monoclonal antibody preparationis directed against a single determinant on an antigen. Thus, themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method.

As used herein, the term “biomarker” shall refer to any molecule orgroup of molecules found in a biological sample that can be used tocharacterize the biological sample or a subject from which thebiological sample is obtained. For example, a biomarker may be amolecule or group of molecules whose presence, absence, or relativeabundance is: characteristic of a particular disease state; indicativeof the severity of a disease or the likelihood of disease progression orregression; and/or predictive that a particular disease state willrespond to a particular treatment. As another example, the biomarker maybe an infectious agent (such as a bacterium, fungus, virus, or othermicroorganism), or a substituent molecule or group of molecules thereof.

As used herein, the terms “sample” and “biological sample” shall referto any composition containing or presumed to contain a biomarker or acomposition being tested for the presence or absence of a particularbiomarker. Samples may include purified or separated components ofcells, tissues, or blood, e.g., DNA, RNA, proteins, cell-free portions,or cell lysates. The sample can be a formalin-fixed, paraffin-embedded(FFPE) tissue sample, e.g., from a tumor or metastatic lesion, e.g.,primary tumor or metastatic tumor. The sample can also be frompreviously frozen or fresh tissue, or from a liquid sample, e.g., bloodor a blood component (plasma or serum), urine, semen, saliva, sputum,mucus, semen, tear, lymph, cerebral spinal fluid, material washed from aswab, etc. Samples also may include constituents and components of invitro cultures of cells obtained from an individual, including celllines. The sample can also be partially processed from a sample directlyobtained from an individual, e.g., cell lysate or blood depleted of redblood cells.

As used herein, the term “cellular sample” refers to any samplecontaining intact cells, such as cell cultures, bodily fluid samples orsurgical specimens taken for pathological, histological, or cytologicalinterpretation.

As used herein, the term “tissue sample” shall refer to a cellularsample that preserves the cross-sectional spatial relationship betweenthe cells as they existed within the subject from which the sample wasobtained. “Tissue sample” shall encompass both primary tissue samples(i.e. cells and tissues produced by the subject) and xenografts (i.e.foreign cellular samples implanted into a subject).

As used herein, “histochemical detection” refers to a process involvinglabeling a biomarker or other structures in a tissue sample withdetection reagents in a manner that permits microscopic detection of thebiomarker or other structures in the context of the cross-sectionalrelationship between the structures of the tissue sample. Examplesinclude affinity histochemistry (AHC), immunohistochemistry (IHC),chromogenic in situ hybridization (CISH), fluorescent in situhybridization (FISH), silver in situ hybridization (SISH), andhematoxylin and eosin (H&E) staining of formalin-fixed,paraffin-embedded tissue sections.

As used herein, “immunohistochemistry” (IHC) refers to a method ofdetermining the presence or distribution of an antigen in a sample bydetecting interaction of the antigen with a specific binding agent, suchas an antibody. A sample is contacted with an antibody under conditionspermitting antibody-antigen binding. Antibody-antigen binding can bedetected by means of a detectable label conjugated to the antibody(direct detection) or by means of a detectable label conjugated to asecondary antibody, which binds specifically to the primary antibody(indirect detection).

Fluorescence (also frequently referred to as immunofluorescence)detection is a widely used IHC technique that can be either direct orindirect: direct indicates that primary antibodies are labeled withfluorescent probes and implies that there is no need to use fluorescentsecondary antibodies, while indirect means that primary antibodies areunlabeled and secondary antibodies conjugated to fluorescent dyes haveto be used for detection. Direct detection is the fastest and shortestIHC protocol, requiring incubation of tissue sections with only aprimary antibody conjugated to the fluorophore of choice. Indirectdetection is more sensitive than direct. The higher sensitivity ofindirect detection is the result of the possibility of two secondaryantibodies labeled with fluorophores interacting with a single moleculeof primary antibody bound to its tissue target. Indirect detectionallows for the ability to choose secondary antibodies with fluorophoresof different colors, Stokes shifts, quantum yield, and fade resistance.Fluorescence detection allows for simultaneous detection of multipleoverlapping and nonoverlapping tissue targets. Multicolor detection canbe done using either direct or indirect detection, or a combination ofboth techniques.

Counterstaining is the staining of tissue sections with dyes that allowone to see the entire “landscape” of the tissue section and serve as areference for the main color used for the detection of tissue targets.Such dyes can stain cell nuclei, the cell membrane, or the entire cell.Examples of dyes include DAPI, which binds to nuclear DNA and emitsstrong blue light; Hoechst blue stain, which binds to nuclear DNA andemits strong blue light; and Propidium iodide, which binds to nuclearDNA and emits strong red light. Counterstaining of the intracellularcytoskeletal network can be done using phalloidin conjugated tofluorescent dyes. Phalloidin is a toxin that tightly binds to actinfilaments in a cell's cytoplasm, which then become clearly visible underthe microscope.

The majority of chromogenic IHC protocols are based on the use of Avidinand Biotin molecules because detection sensitivity of a simpleantigen-antibody reaction in many cases is quite low. Avidin-Biotinbinding serves to bridge antigen-bound antibodies with detectionreagents, allowing amplification of the staining signal. The mostfrequently used Biotin-based techniques include labeled SA-Biotin (LSAB)and Avidin-Biotin Complex (ABC) detection. There are alsonon-Biotin-based detection techniques utilizing primary antibodieseither conjugated directly to enzymatic labels or to a long polymercontaining multiple copies of enzymatic labels.

LSAB Detection utilizes secondary antibodies conjugated to Biotin thatlink primary antigen-bound antibodies to SA conjugated to an enzyme. Thefirst step in LSAB detection is the incubation of tissue sections withprimary antibodies followed by incubation with biotinylated secondaryantibodies. After that, SA conjugated to the enzyme of choice (e.g., AP,HRP, etc.) is added to tissue sections followed by adding appropriateenzyme substrate. The enzyme converts substrate into colored particlesprecipitating at the sites of antigen localization, which can then beobserved under the microscope. LSAB technique can be shortened usingbiotinylated primary antibodies, eliminating the need for incubationwith biotinylated secondary antibodies.

The initial steps—incubation with primary and biotinylated secondaryantibodies—in ABC detection are the same as in LSAB, but the next stepsand reagents are quite different. Avidin and biotinylated enzymes arefirst mixed and incubated together for about 30 min at room temperatureand then added to tissue sections. During this incubation, Avidininteracts with the biotinylated enzymes, forming large complexes denselypacked with enzyme molecules—far exceeding the concentration found inthe LSAB detection technique—that boost the sensitivity of antigendetection.

Non-Biotin detection techniques have gained popularity because they aredevoid of such limitations of Avidin-Biotin detection as nonspecificbackground staining due to the endogenous biotin that is abundant indifferent types of animal tissues, including kidney, brain, andplacenta.

In chromogenic IHC, tissue counterstaining serves the same purpose as itdoes in fluorescence detection: to visualize the entire layout of thetissue section and label organelles of the same type. Usuallycounterstaining is done to label cell nuclei that should not be of thesame color as the main color depicting antigens of interest. Forexample, if the main color is red (AEC chromogen) or brown (DABchromogen), nuclei may be stained using Hematoxylin, which produces ablue color, or Methyl Green, which produces a green color. If the maincolor is blue (BCIP/NBT chromogen), then nuclei may be counterstainedred using Nuclear Fast Red dye. In cases when tissue antigen islocalized in cell nuclei, the duration of their counterstaining may beeither shortened to make them barely visible or even skipped to avoidmasking the main IHC color.

AHC refers to affinity histochemistry wherein detection of a biomarkerinvolves the use of a binding agent with affinity for the biomarker. Forexample, mast cells may be stained by AHC based on electrostaticattractions between the basic protein avidin and the polyanionic heparin(identifiable by immunofluorescence).

As used herein, the term “section” shall refer to a thin slice of atissue sample suitable for microscopic analysis, typically cut using amicrotome. As an example, a section may be 4 to 5 microns thick. Thepresent invention is not limited to 4 to 5 microns.

As used herein, the term “serial section” shall refer to any one of aseries of sections cut in sequence from a tissue sample. For twosections to be considered “serial sections” of one another, they do notnecessarily need to consecutive sections from the tissue, but theyshould generally contain the same tissue structures in the samecross-sectional relationship, such that the structures can be matched toone another after histological staining.

As used herein, the phrase “specific binding,” “specifically binds to,”or “specific for” refers to measurable and reproducible interactionssuch as binding between a target and a biomarker-specific agent, whichis determinative of the presence of the target in the presence of aheterogeneous population of molecules including biological molecules.For example, a binding entity that specifically binds to a target is anantibody that binds this target with greater affinity, avidity, morereadily, and/or with greater duration than it binds to other targets.

As used herein, the term “biomarker-specific agent” or “specific bindingagent” shall refer to any compound or composition that binds to abiomarker or a specific structure within that biomarker in a manner thatpermits a specific detection of the biomarker in a sample. Examplesinclude: antibodies and antigen binding fragments thereof; andengineered specific binding structures, including ADNECTINs (scaffoldbased on 10th FN3 fibronectin; Bristol-Myers-Squibb Co.), AFFIBODYs(scaffold based on Z domain of protein A from S. aureus; Affibody AB,Solna, Sweden), AVIMERs (scaffold based on domain A/LDL receptor; Amgen,Thousand Oaks, Calif.), dAbs (scaffold based on VH or VL antibodydomain; GlaxoSmithKline PLC, Cambridge, UK), DARPins (scaffold based onAnkyrin repeat proteins; Molecular Partners AG, Zurich, CH), ANTICALINs(scaffold based on lipocalins; Pieris AG, Freising, Del.), NANOBODYs(scaffold based on VHH (camelid Ig); Ablynx N/V, Ghent, BE), TRANS-BODYs(scaffold based on Transferrin; Pfizer Inc., New York, N.Y.), SMIPs(Emergent Biosolutions, Inc., Rockville, Md.), and TETRANECTINs(scaffold based on C-type lectin domain (CTLD), tetranectin; BoreanPharma A/S, Aarhus, DK) (Descriptions of such engineered specificbinding structures are reviewed by Wurch et al., Development of NovelProtein Scaffolds as Alternatives to Whole Antibodies for Imaging andTherapy: Status on Discovery Research and Clinical Validation, CurrentPharmaceutical Biotechnology, Vol. 9, pp. 502-509 (2008), the content ofwhich is incorporated by reference); and fusion proteins including atleast a first domain capable of specifically binding to the biomarker(e.g. an antigen binding fragment of an antibody or a target-bindingportion of a protein that binds to the biomarker) and a second portionthat is adapted to facilitate binding of detection reagents to thefusion protein (e.g., a biotin label, an epitope tag, an Ig fragment,etc.).

A “detection reagent” when used in connection with a histochemical assay(including immunohistochemistry and affinity histochemistry) is anyreagent that is used to deposit a stain in proximity to abiomarker-specific agent bound to a biomarker in a cellular sample.Non-limiting examples include secondary antibodies capable of binding toa biomarker-specific antibody; enzymes linked to such secondaryantibodies; and chemicals reactive with such enzymes to effectdeposition of a fluorescent or chromogenic stain; and the like.

When used as a noun, the term “stain” shall refer to any substance thatcan be used to visualize specific molecules or structures in a cellularsample for microscopic analysis, including bright field microscopy,fluorescent microscopy, electron microscopy, and the like. When used asa verb, the term “stain” shall refer to any process that results indeposition of a stain on a cellular sample (e.g., tissue sample,cytological sample, etc.).

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULARBIOLOGY, Elsevier (4th ed. 2007); Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, N.Y.1989). The term “a” or “an” is intended to mean “one or more.” The terms“comprise,” “comprises,” and “comprising,” when preceding the recitationof a step or an element, are intended to mean that the addition offurther steps or elements is optional and not excluded.

Systems and Applications for the Present Invention

The present invention features compositions comprising a solid,dissolvable reagent composition for delivering said reagent (e.g.,antibodies, probes, buffers, chromogens, counterstains, etc.) to asample. The present invention also features methods of producing saidcompositions, methods (applications) of using said compositions (e.g.,methods of introducing antibodies to a sample, etc.), and systems (e.g.,automated systems) featuring the use of said compositions. For example,the present invention features methods, systems, and compositions fordelivering a reagent to a sample, e.g., delivering an antibody to atissue sample or tissue section adhered to a slide (e.g., for thedetection of a biomarker).

In some embodiments, the systems or compositions of the presentinvention are used for histochemical applications, e.g., the delivery ofantibodies or probes or other reagents to a tissue section, automatedhistochemical staining methods as described herein, etc. The presentinvention is not limited to histochemical applications. For example, insome embodiments, the systems or compositions of the present inventionare used for other tests or assays, such as ELISAs, staining assays(e.g., for primary stains, special stains), or any other method or testor assay involving the application or a reagent such as a detectionreagent, a binding reagent, a biomarker-specific agent, an enzyme, aprotein, a nucleic acid, a stain, etc., wherein the reagent can beapplied as a solid, dissolvable reagent.

In some embodiments, the systems or compositions of the presentinvention are used in manual methods. In some embodiments, the systemsor compositions of the present invention are used in automated methods.

Automated Staining Machines

The solid, dissolvable reagents of the present invention may be used inan automated staining machine. The methods of the present invention maybe performed on an automated staining machine (slide stainer) or otherappropriate automated slide processing machine, e.g., a slide stainingmachine that utilizes puddle technology to deliver reagents. Specificexamples of automated staining machines (e.g., IHC/ISH slide stainers)include: itelliPATH (Biocare Medical), WAVE (Celerus Diagnostics), DAKOOMNIS and DAKO AUTOSTAINER LINK 48 (Agilent Technologies), BENCHMARK XT(Ventana Medical Systems, Inc.), BENCHMARK ULTRA (Ventana MedicalSystems, Inc.), BENCHMARK GX (Ventana Medical Systems, Inc.), VENTANAH&E 600 (Ventana Medical Systems, Inc.), BENCHMARK Special Stains(Ventana Medical Systems, Inc.), VENTANA DISCOVERY XT (Ventana MedicalSystems, Inc.), VENTANA DISCOVERY ULTRA (Ventana Medical Systems, Inc.),Leica BOND, and Lab Vision Autostainer (Thermo Scientific). Automatedstaining machines (automated slide stainers) are also described byPrichard, Overview of Automated Immunohistochemistry, Arch Pathol LabMed., Vol. 138, pp. 1578-1582 (2014), incorporated herein by referencein its entirety. Additionally, Ventana Medical Systems, Inc. is theassignee of a number of United States patents disclosing systems andmethods for performing automated analyses, including U.S. Pat. Nos.5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901 and 6,943,029, andU.S. Published Patent Application Nos. 20030211630 and 20040052685, eachof which is incorporated herein by reference in its entirety. Themethods of the present invention may be adapted to be performed on anyappropriate automated staining machine (or automated slide processingmachine).

Automated IHC/ISH slide stainers typically include at least a stainerunit for dispensing reagent to implement staining protocols onto aslide. Commercially-available staining units typically operate on one ofthe following principles: (1) open individual slide staining, in whichslides are positioned horizontally and reagents are dispensed as apuddle on the surface of the slide containing a tissue sample (such asimplemented on the DAKO AUTOSTAINER Link 48 (Agilent Technologies) andintelliPATH (Biocare Medical) stainers); (2) liquid overlay technology,in which reagents are either covered with or dispensed through an inertfluid layer deposited over the sample (such as implemented on VENTANABenchMark and DISCOVERY stainers); (3) capillary gap staining, in whichthe slide surface is placed in proximity parallel to another surface(which may be another slide or a coverplate) to create a narrow gap,through which capillary forces draw up and keep liquid reagents incontact with the samples (such as the staining principles used by DAKOTECHMATE, Leica BOND, and DAKO OMNIS stainers). Some iterations ofcapillary gap staining do not mix the fluids in the gap (such as on theDAKO TECHMATE and the Leica BOND). In some variations of capillary gapstaining, the reagents are mixed in the gap, such as translating gaptechnology, in which a gap is created between the slide and a curvedsurface and movement of the surfaces relative to one another effectsmixing (see U.S. Pat. No. 7,820,381); and dynamic gap staining, whichuses capillary forces similar to capillary gap staining to apply sampleto the slide, and then translates the parallel surfaces relative to oneanother to agitate the reagents during incubation to effect reagentmixing (such as the staining principles implemented on DAKO OMNIS slidestainers (Agilent)). It has recently been proposed to use inkjettechnology to deposit reagents on slides. See WO 2016-170008 A1. As anexample, the VENTANA DISCOVERY ULTRA system features slide drawers with30 independent slide reaction chambers with dedicated bulk reagentsupply lines and individual slide heaters, a reagent carousel with 35reagent positions, and the ability to hold up to 7 different bulkreagents in 3- to 6-liter onboard containers. The system has a slidetemperature range from ambient temperature up to about 100° C. TheBENCHMARK Special Stains system has a slide carousel for processing upto 20 slides with independent temperature control for each position, areagent carousel with 25 reagents positions, and the ability to hold upto 4 bulk solutions in 3- to 6-liter onboard containers. The BenchMarkULTRA system can process up to 30 slides with independentprocessing/functionality and temperature control for each position. Thesystem has a reagent carousel with 35 reagent positions and the abilityto hold up to 7 different bulk reagents, which can be changed withoutprocess interruption. The system has a slide temperature range fromambient temperature up to about 100° C.

This list of staining principles is not intended to be exhaustive, andthe present methods and systems are intended to include any stainingtechnology (both known and to be developed in the future) that can beused to apply the appropriate reagents to the sample.

Samples

As previously discussed, the sample used in the methods and systems ofthe present invention may be compositions comprising cells or tissue, orpurified or separated components of cells, tissues, or blood, e.g., DNA,RNA, proteins, cell-free portions, or cell lysates. The sample may be aformalin-fixed, paraffin-embedded (FFPE) tissue sample, e.g., from atumor or metastatic lesion, e.g., primary tumor or metastatic tumor, ahealthy sample, etc. The sample can also be from previously frozen orfresh tissue, or from a liquid sample, e.g., blood or a blood component(plasma or serum), urine, semen, saliva, sputum, mucus, semen, tear,lymph, cerebral spinal fluid, material washed from a swab, etc. In someembodiments, the sample in vitro cultures of cells obtained from anindividual, including cell lines. In some embodiments, the sample ispartially processed from a sample directly obtained from an individual,e.g., cell lysate or blood depleted of red blood cells.

In some embodiments, the samples used in the present invention aretissue samples or tissue sections, such as tissue sections adhered to aslide. However, the present invention is not limited to tissue samples,and the present invention is not limited to samples adhered to a slide.For example, in some embodiments, the sample is in a tube, a well (e.g.,a multiwell plate), or other container. In some embodiments, the sampleis a solid sample. In some embodiments, the sample is a liquid-basedsample. In some embodiments, the sample comprises tissue or cells. Insome embodiments, the sample comprises nucleic acid, protein, bacteria,viruses, or any other test agent.

As an example, a tissue sample may be of any type appropriate forhistological examination on an automated staining machine (as describedherein). The tissue sample may comprise a biopsy or surgical resection.The tissue sample may comprise a section of cancerous and/or healthytissue. In some embodiments, the sample is fresh, frozen, formalin fixedparaffin embedded (FFPE), etc. In some embodiments, the tissue sample isfixed in order to preserve the shape of the cells or tissue. Commonfixatives include formaldehyde, ethanol, methanol, and/or picric acid.

If the tissue sample is a sample embedded in paraffin, the sample can bedeparaffinized with the automated IHC/ISH slide stainer usingappropriate deparaffinizing fluid(s). After the waste remover removesthe deparaffinizing fluid(s), any number of substances can besuccessively applied to the sample. The substances can be forpretreatment, cell lysis, denaturation, washing or the like.

Reagents

Reagents for immunohistochemistry or in situ hybridization includeantibodies, buffers, stains, nucleic acids, enzymes, as well as othermolecules such as tyramide or other detection reagents. The solidcompositions (water-soluble polymer compositions) of present inventionmay include any of the aforementioned reagents. Non-limiting examples ofantibodies (Ventana Medical Systems, Inc.) include: Estrogen Receptor(ER) (SP1) rabbit monoclonal primary antibody; EGFR (5B7) rabbitmonoclonal primary antibody; FITC IgG primary antibody; c-MYC (Y69)rabbit monoclonal primary antibody; CD8 (SP57) rabbit monoclonal primaryantibody, etc. Non-limiting examples of nucleic acids (Ventana MedicalSystems, Inc.) includes: Chromosome 3 DIG probe; Chromosome 17 DIGprobe; EGFR DNP Probe; HER2 Dual ISH DNA Probe cocktail; HPV6 mRNAprobe; Kappa DNP probe; Lambda DNP probe; MYC DNP probe; etc.Non-limiting examples of other reagents (Ventana Medical Systems, Inc.)include: ISH Protease 1; Protease 3; hematoxylin; bluing reagent;hematoxylin II; blocking reagents; amplification reagents; etc.

Representative buffering agents or salts include, but are not limitedto, Tris, Tricine, HEPES, MOPS, TAPS, Bicine, TAPSO, TES, PIPES,Cacodylate, SSC, MES, KCl, NaCl, potassium acetate, NH4-acetate,potassium glutamate, NH4Cl, ammonium sulphate, MgCl2, magnesium acetateand the like. Other agents that may be present in the buffer mediuminclude chelating agents, such as EDTA, EGTA and the like.

Non-limiting examples of chromogen substrates that may be used in thesolid reagents of the present invention include but are not limited toDAB, AEC, CN, BCIP/NBT, FAST RED, FAST BLUE, FUCHSIN, NBT, ALK, See alsoU.S. Pat. Publ. 2013/0260379 which is incorporated by reference hereinin its entirety.

The aforementioned reagents are for exemplary purposes only and are notintended to limit the present invention to those reagents.

Solid Reagent Compositions

Any appropriate configuration of the solid reagent composition may beconsidered. While the present disclosure mentions solid reagentcompositions in the form of wafers or films (a reagent embedded inwafers or films), packets (e.g., packets filled with a reagent), orreagent-printed films (e.g., a reagent inkjet printed onto films), thepresent invention is not limited to these configurations. Further, thesolid reagent compositions of the present invention may be constructedfrom a variety of materials.

A. Films or Wafers

In certain embodiments, the solid reagent composition is in the form ofa wafer or film, wherein the wafer or film comprises a water-solublepolymer and one or more reagents embedded therein. In some embodiments,the water-soluble polymer that makes up the wafer or film comprisespolyvinyl alcohol (PVA), however the present invention is not limited toPVA. For example, in some embodiments, the water-soluble polymercomprises dextran, hydroxypropyl cellulose, poly (acrylic acid sodiumsalt), poly (ethylene glycol), poly (methylacrylic acid sodium salt),poly(styrenesulfonic acid sodium salt), pullulan, maltodextrin,microcrystalline cellulose, maltodextrin, microcrystalline cellulose,PVA, the like, or a combination thereof. PVA is well known to one ofordinary skill in the art.

In certain embodiments, the water-soluble polymer further comprises aplasticizer, e.g., a formulation that helps improve flexibility of thefilm or wafer. Non-limiting examples of materials used as plasticizersinclude polytheylene glycols, glycerol, low moleculer weightpolyethylene glycols, phthalate derivatives (e.g., dimethyl phthalate,diethyl phthalate, dibutyl phthalate), citrate derivatives (e.g.,tributyl citrate, triethyl citrate, acetyl citrate), triacetin, castoroil, etc.

Reagent films or wafers (e.g., PVA films, PVA wafers, etc.) may beconstructed by dissolving water-soluble polymer (e.g., PVA) powder in asolvent. Non-limiting examples of solvents include ethanol-deionized(DI) water solutions (e.g., 50% ethanol-50% DI water, 60% ethanol-40% DIwater, etc.), the like, or buffers, e.g., BenchMark ULTRA ReactionBuffer. Without wishing to limit the present invention to any theory ormechanism, it is believed that a buffer may be more beneficial forcertain applications because it is safer to heat in order to dissolvethe water-soluble polymer (e.g., PVA) quickly, and it also provides astable environment for antibodies.

In certain embodiments, PVA wafers or films are constructed using PVAsolutions (w/v). In certain embodiments, the PVA solution comprises 0.1%PVA (w/v). In certain embodiments, the PVA solution comprises 0.5% PVA(w/v). In certain embodiments, the PVA solution comprises 1% PVA (w/v).In certain embodiments, the PVA solution comprises 2% PVA (w/v). Incertain embodiments, the PVA solution comprises 3% PVA (w/v). In certainembodiments, the PVA solution comprises 4% PVA (w/v). In certainembodiments, the PVA solution comprises 5% PVA (w/v). In certainembodiments, the PVA solution comprises 6% PVA (w/v). In certainembodiments, the PVA solution comprises 7% PVA (w/v). In certainembodiments, the PVA solution comprises 8% PVA (w/v). In certainembodiments, the PVA solution comprises 9% PVA (w/v). In certainembodiments, the PVA solution comprises 10% PVA (w/v). In certainembodiments, the PVA solution comprises from 0.1 to 1% PVA (w/v). Incertain embodiments, the PVA solution comprises from 1 to 5% PVA (w/v).In certain embodiments, the PVA solution comprises from 0.1 to 10% PVA(w/v). In certain embodiments, the PVA solution comprises from 0.5 to 5%PVA (w/v). In certain embodiments, the PVA solution comprises from 2 to5% PVA (w/v). In certain embodiments, the PVA solution comprises from 2to 10% PVA (w/v).

The reagent or reagents are added to the water-soluble polymer (e.g.,PVA) solutions. Subsequently, the reagent-water-soluble polymer (e.g.,PVA) solutions are dehydrated, resulting in a reagent-embeddedwater-soluble polymer (e.g., PVA) film. In certain embodiments,water-soluble polymer (e.g., PVA) solutions are dehydrated at 37° C.,38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., etc. fora certain length of time.

B. Packets

In certain embodiments, the water-soluble polymer is in the form of apacket, wherein the packet comprises a water-soluble polymer with one ormore reagents contained therein. In some embodiments, the water-solublepolymer that forms the packet comprises polyvinyl alcohol (PVA), howeverthe present invention is not limited to PVA. For example, in someembodiments, the water-soluble polymer comprises dextran, hydroxypropylcellulose, poly (acrylic acid sodium salt), poly (ethylene glycol), poly(methylacrylic acid sodium salt), poly(styrenesulfonic acid sodiumsalt), pullulan, maltodextrin, microcrystalline cellulose, PVA, thelike, or a combination thereof. PVA is well known to one of ordinaryskill in the art.

In certain embodiments, the water-soluble polymer further comprises aplasticizer, e.g., a formulation that helps improve flexibility of thefilm or wafer. Non-limiting examples of materials used as plasticizersinclude polytheylene glycols, glycerol, low moleculer weightpolyethylene glycols, phthalate derivatives (e.g., dimethyl phthalate,diethyl phthalate, dibutyl phthalate), citrate derivatives (e.g.,tributyl citrate, triethyl citrate, acetyl citrate), triacetin, castoroil, etc.

In certain embodiments, pre-made films of water-soluble polymer (e.g.,PVA films) are used to create the packet. In certain embodiments, filmsof the water-soluble polymer (e.g., PVA films) are custom made.

As previously discussed, films (e.g., PVA films) may be constructed bydissolving water-soluble polymer (e.g., PVA) powder in a solvent.Non-limiting examples of solvents include ethanol-deionized (DI) watersolutions (e.g., 50% ethanol-50% DI water, 60% ethanol-40% DI water,etc.), the like, or buffers, e.g., BenchMark ULTRA Reaction Buffer. Asan example, in certain embodiments, PVA films are constructed using PVAsolutions (w/v). In certain embodiments, the PVA solution comprises 0.1%PVA (w/v). In certain embodiments, the PVA solution comprises 0.5% PVA(w/v). In certain embodiments, the PVA solution comprises 1% PVA (w/v).In certain embodiments, the PVA solution comprises 2% PVA (w/v). Incertain embodiments, the PVA solution comprises 3% PVA (w/v). In certainembodiments, the PVA solution comprises 4% PVA (w/v). In certainembodiments, the PVA solution comprises 5% PVA (w/v). In certainembodiments, the PVA solution comprises 6% PVA (w/v). In certainembodiments, the PVA solution comprises 7% PVA (w/v). In certainembodiments, the PVA solution comprises 8% PVA (w/v). In certainembodiments, the PVA solution comprises 9% PVA (w/v). In certainembodiments, the PVA solution comprises 10% PVA (w/v). In certainembodiments, the PVA solution comprises from 0.1 to 1% PVA (w/v). Incertain embodiments, the PVA solution comprises from 1 to 5% PVA (w/v).In certain embodiments, the PVA solution comprises from 0.1 to 10% PVA(w/v). In certain embodiments, the PVA solution comprises from 0.5 to 5%PVA (w/v). In certain embodiments, the PVA solution comprises from 2 to5% PVA (w/v). In certain embodiments, the PVA solution comprises from 2to 10% PVA (w/v).

The water-soluble polymer (e.g., PVA) solutions are then dehydrated,resulting in a water-soluble polymer film (e.g., PVA film). In certainembodiments, water-soluble polymer (e.g., PVA) solutions are dehydratedat 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45°C., etc. for a certain length of time.

The water-soluble polymer films (e.g., PVA films), whether custom madeor pre-made, are configured to form an inner compartment for storing thereagent therein. For example, two (or more) water-soluble polymer films(e.g., PVA films) are melted together to form a packet with an openingfor insertion of the reagent. In certain embodiments, several layers ofwater-soluble polymer films are stacked to form a portion (e.g., a side)of the packet. The reagent may be diluted in a solution that does notdissolve the water-soluble polymer. For example, in certain embodiments,the reagent (e.g., antibody) is diluted in glycerin. Once the reagentsolution is added to the inner compartment of the packet, the packet issealed (e.g., with heat).

C. Inkjet Printed Films

In certain embodiments, the water-soluble polymer is in the form of aprinted film (e.g., inkjet printed film), wherein the film comprises awater-soluble polymer with one or more reagents printed thereon.

In some embodiments, the film comprises polyvinyl alcohol (PVA), howeverthe present invention is not limited to PVA. For example, in someembodiments, the water-soluble polymer comprises dextran, hydroxypropylcellulose, poly (acrylic acid sodium salt), poly (ethylene glycol), poly(methylacrylic acid sodium salt), poly(styrenesulfonic acid sodiumsalt), pullulan, PVA, the like, or a combination thereof. PVA is wellknown to one of ordinary skill in the art.

In certain embodiments, the water-soluble polymer further comprises aplasticizer, e.g., a formulation that helps improve flexibility of thefilm or wafer. Non-limiting examples of materials used as plasticizersinclude polytheylene glycols, glycerol, low moleculer weightpolyethylene glycols, phthalate derivatives (e.g., dimethyl phthalate,diethyl phthalate, dibutyl phthalate), citrate derivatives (e.g.,tributyl citrate, triethyl citrate, acetyl citrate), triacetin, castoroil, etc.

In certain embodiments, pre-made films of water-soluble polymer (e.g.,PVA films) are used for printing the reagent thereon. In certainembodiments, films of the water-soluble polymer (e.g., PVA films) arecustom made.

As previously discussed, films (e.g., PVA films) may be constructed bydissolving water-soluble polymer (e.g., PVA) powder in a solvent.Non-limiting examples of solvents include ethanol-deionized (DI) watersolutions (e.g., 50% ethanol-50% DI water, 60% ethanol-40% DI water,etc.), the like, or buffers, e.g., BenchMark ULTRA Reaction Buffer. Asan example, in certain embodiments, PVA films are constructed using PVAsolutions (w/v). In certain embodiments, the PVA solution comprises 0.1%PVA (w/v). In certain embodiments, the PVA solution comprises 0.5% PVA(w/v). In certain embodiments, the PVA solution comprises 1% PVA (w/v).In certain embodiments, the PVA solution comprises 2% PVA (w/v). Incertain embodiments, the PVA solution comprises 3% PVA (w/v). In certainembodiments, the PVA solution comprises 4% PVA (w/v). In certainembodiments, the PVA solution comprises 5% PVA (w/v). In certainembodiments, the PVA solution comprises 6% PVA (w/v). In certainembodiments, the PVA solution comprises 7% PVA (w/v). In certainembodiments, the PVA solution comprises 8% PVA (w/v). In certainembodiments, the PVA solution comprises 9% PVA (w/v). In certainembodiments, the PVA solution comprises 10% PVA (w/v). In certainembodiments, the PVA solution comprises from 0.1 to 1% PVA (w/v). Incertain embodiments, the PVA solution comprises from 1 to 5% PVA (w/v).In certain embodiments, the PVA solution comprises from 0.1 to 10% PVA(w/v). In certain embodiments, the PVA solution comprises from 0.5 to 5%PVA (w/v). In certain embodiments, the PVA solution comprises from 2 to5% PVA (w/v). In certain embodiments, the PVA solution comprises from 2to 10% PVA (w/v).

The water-soluble polymer (e.g., PVA) solutions are then dehydrated,resulting in a water-soluble polymer film (e.g., PVA film). In certainembodiments, water-soluble polymer (e.g., PVA) solutions are dehydratedat 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45°C., etc. for a certain length of time.

The water-soluble polymer films (e.g., PVA films), whether custom madeor pre-made, are used for printing one or more reagents thereon. Incertain embodiments, inkjet printing is used to print the reagent(s) onthe films. For example, solid water-soluble polymer films can be cut andplaced onto an inject printer platform for printing. The presentinvention is not limited to inkjet printing.

Without wishing to limit the present invention to any theory ormechanism, it is believed that since the reagent is printed, theinkjet-printed antibody film does not require a particular liquiddiluent (e.g., glycerin) to carry the antibody within the PVA. Further,the printing can be accomplished on just a single layer of water-solublepolymer (e.g., PVA), as opposed to two or more layers. A single layer ofpolymer will dissolve more readily during the assay.

EXAMPLES Example 1—Process of Making Reagent-Embedded Wafers

Example 1 describes the production of several solid wafer reagents,e.g., one comprising Hematoxylin II, one comprising Bluing Reagent, etc.The present invention is not limited to the methods, compositions, andconfigurations described in Example 1.

PVA wafers were initially made by preparing a 2% or 5% PVA solution bydissolving PVA powder (M_(w) 89,000-98,000, 99+% hydrolyzed; SigmaAldrich, P/N 341584) in 50% ethanol, and 50% deionized (DI) water.Additional wafers were made by dissolving PVA in BenchMark UltraReaction Buffer (P/N 950-300), which helped provide a stable environmentfor antibodies. For experiments described in this example, 2 g or 5 g ofPVA powder, for 2% and 5% PVA solutions (w/v), respectively, was mixedwith 100 mL of Reaction Buffer in a beaker, and stirred with a stir barat 90-100° C. to facilitate dissolution until the solution was clear.400 μL of each PVA solution was pipetted onto a clean polystyrene orceramic tray to form a puddle, and a single slide dispense (100 μL) ofeach OptiView IHC detection kit (e.g., PTEN (SP218) Rabbit MonoclonalPrimary Antibody (P/N 790-5097, Ventana Medical Systems, Tucson, Ariz.),Hematoxylin II (P/N 790-2208, Ventana Medical Systems, Tucson, Ariz.),or Bluing Reagent (P/N 760-2037, Ventana Medical Systems, Tucson,Ariz.)) or ancillary reagent being tested was dispensed directly ontothe puddle of PVA solution (1×). The reagent-PVA solution was thendehydrated at 37° C. for 1-2 hours, resulting in a reagent-embedded PVAfilm (see FIG. 1A). FIG. 1B shows the wafer dissolved when applied tothe reaction buffer puddle on a slide.

Example 2—Process of Making Antibody-Embedded Wafers

Example 2 describes the production of a solid reagent comprising anantibody, wherein the antibody is embedded into a wafer. The presentinvention is not limited to the methods, compositions, andconfigurations described in Example 2. For example, the presentinvention is not limited to embedding a reagent into a wafer or film,the use of an antibody, the use of PVA, etc.

1-4% PVA solutions were made from PVA powder (Mw 89,000-98,000, 99+%hydrolyzed; Sigma Aldrich, P/N 341584), ethanol, and deionized (DI)water. 1-4 g of PVA powder, for 1% and 4% PVA solutions (w/v),respectively, was mixed with 40 mL of DI water and 60 mL of 100% ethanolin a beaker, and stirred with a stir bar at 80-90° C. to facilitatedissolution until the solution was clear. 400 μL of each PVA solutionwas pipetted into a clean tray to form a puddle, and a single slidedispense (100 μL) of PTEN (SP218) Rabbit Monoclonal Primary Antibody(P/N 790-5097, Ventana Medical Systems, Tucson, Ariz.) was dispenseddirectly onto puddle of PVA solution (1×). The antibody-PVA solution wasthen dehydrated at 45° C. resulted in an antibody embedded PVA film.FIG. 2A shows the antibody-PVA solution as distributed in trays in 500μL droplets for dehydration into a film in an oven at 45° C. FIG. 2Bshows a VENTANA BenchMark ULTRA automated stainer wherein the primaryantibody hand titration option in the staining procedure is used toapply the solid reagent. Purple food coloring was added to the PVA forvisualization purposes.

Example 3—Process of Making Antibody Packet

Example 3 describes the production of a solid reagent comprising anantibody, wherein a packet is filled with an antibody-glycerin solution.The present invention is not limited to the methods, compositions, andconfigurations described in Example 3. For example, the presentinvention is not limited to the use of antibodies, the use of PVA, apacket configuration, etc.

A NSCLC sample slide was prepared according to standard protocols asknown to one of skill in the art. Commercially available PVA film waspurchased (SULKY of America; Item No. 486-12). The PVA film was cut intosmall squares approximately 4 cm×4 cm with scissors. Two squares werestacked and melted together with a soldering iron to form ¾ of a packetoutline. A layer of wax paper was used to transfer heat. Raw PTEN(SP218) Rabbit Monoclonal Primary Antibody was diluted 1× (1:300) inglycerin (which would not dissolve the water soluble PVA). 100 μL of theantibody-glycerin solution was pipetted into the PVA packet. The packetwas sealed closed with the soldering iron (see FIG. 3A). Red foodcoloring was added for visualization.

Referring to FIG. 3B, the PVA packet filled with antibody diluted inglycerin (of FIG. 3A) was placed on slides on the VENTANA BenchMarkULTRA automated stainer (Ventana Medical Systems, Tucson, Ariz.) usingthe primary antibody hand titration option in the staining procedure.The puddle of reaction buffer on top of the slide dissolved the PVA,releasing the antibody and enabling staining with the primary antibody.Red food coloring was added for visualization.

Example 4—Process of Making Inkjet Printed Antibody Film

Example 4 describes the production of a solid reagent comprising anantibody, wherein the antibody is printed onto a film. The presentinvention is not limited to the methods, compositions, andconfigurations described in Example 4. For example, the presentinvention is not limited to manufacturing methods using printing, theuse of an antibody, the use of PVA, etc.

Commercially available PVA film was purchased (SULKY of America; ItemNo. 486-12). PVA film was cut into small squares approximately 4 cm×4cm, and placed onto the inkjet printer platform (FIG. 4A). Raw CD34(QBEnd/10) Mouse Monoclonal Primary Antibody was diluted 200× (1:30) inReaction Buffer, Glycerol, Tris-HCl and Brij-35 to simulate the antibodydiluent. The antibody solution was loaded into inkjet cartridges and wasprinted onto the PVA film in a 1 in.×0.5 in. area at print density of300 dpi to match the mass of antibody (1×) in a single slide dispense(FIG. 4B).

Example 5—Experimental Testing and Results for Reagent-Embedded Wafers

Example 5 describes testing of the reagent-embedded wafers of Example 1.The present invention is not limited to the methods, systems, andcompositions described herein.

A. Procedure for Immunohistochemistry (IHC) Staining

Wafers made for each of the OptiView IHC and counterstain reagents (seeExample 1) tested were stored at room temperature for less than 2 daysprior to the functional testing with IHC staining runs. A customizedstaining procedure was created based on the staining procedure, “UOptiView DAB IHC v5.2 CDx” on a BenchMark ULTRA using the selectionslisted in Table 1 for the PTEN (SP218) primary antibody and NegativeReagent Control. The staining procedure was altered to allow for manualapplication “hand-apply” steps for the manual application of the reagentwafers being tested.

TABLE 1 Staining protocol selections used for functional testing of theprototypes on the BenchMark ULTRA using the U OptiView DAB IHC v5.2 CDxprocedure Negative Reagent Procedure Step PTEN (SP218) ControlDeparaffinization Selected Selected Cell Conditioning (CC1) 100° C., 56minutes 100° C., 56 minutes Pre antibody peroxidase Selected Selectedinhibitor Antibody Incubation 16 minutes, 37° C. 16 minutes, 37° C.OptiView HQ Linker 8 minutes (default) 8 minutes (default) OptiView HRPMultimer 8 minutes (default) 8 minutes (default) Hematoxylin II 4minutes 4 minutes Bluing Reagent 4 minutes 4 minutes

Referring to Table 1, unstained slides of normal human pancreas tissueswere placed in BenchMark ULTRA instruments and stained with PTEN (SP218)rabbit monoclonal primary antibody or rabbit monoclonal negative controlIg. The PVA film prototypes were placed manually onto the slide duringthe “hand apply antibody titration” step, where the instrument allowsthe user to open the slide drawer to manually apply antibody into thereaction buffer puddle on top of the slide. Controls for each run wereincluded, where a normal dispenser was used to dispense the antibodyonto the slide instead of the PVA prototype. Reaction buffer was alsopipetted on top of the PVA film (approximately 400 μL) in order tocompensate for buffer displaced by placement of the prototype, and tospeed up PVA dissolution. Although this reaction buffer step wasperformed manually, it could be added to the staining procedure bychanging the hand apply software macro to include a reaction buffer“adjust” step after closing the slide drawer. After each staining run,the slides were dehydrated and coverslipped according to the OptiViewDAB Detection Kit package insert (1010323EN).

B. Results for Reagent-Embedded Wafer Compositions

Referring to FIG. 5, FIG. 6, FIG. 7, and FIG. 8, reagent-embedded PVAfilm wafers were used to stain normal pancreas tissues with PTEN (SP218)rabbit monoclonal antibody. Each detection reagent tested (Linker,Multimer, DAB, Copper, Hematoxylin and Bluing Reagent) was testedindividually and then ultimately combined for an all-wafer staining run(see FIG. 8). Each run resulted in successful DAB detection of the PTEN(SP218) antibody, and counterstain. The staining results were consistentto the dispenser controls for the Linker, Multimer, DAB and Copperwafers (see FIG. 5, FIG. 6). Although Bluing Reagent and Hematoxylinwafers did result in successfully staining the tissue with counterstain,the staining quality was not comparable to the dispenser controls.Further optimization is needed to obtain acceptable staining quality(see FIG. 7).

Example 6—Experimental Testing and Results for Antibody-Embedded Wafers,Antibody Packets, and Inkjet Printed Antibody Films

Example 6 describes testing of the solid reagents, e.g.,antibody-embedded wafers, antibody packets, and inkjet printed antibodyfilms, of Example 2, Example 3, and Example 4, respectively. The presentinvention is not limited to the methods, systems, and compositionsdescribed herein.

A. Procedure for Immunohistochemistry (IHC) Staining

Each of the three prototypes described in Example 2, Example 3, andExample 4 were stored at 4° C. for less than 5 days prior to thefunctional testing with IHC staining runs. Staining protocols werecreated using the staining procedure, “U OptiView DAB IHC v5.2 CDx” on aBenchMark ULTRA automated stainer using the selections listed in Table 1for the PTEN (SP218) and CD34 primary antibodies.

TABLE 2 Staining protocol selections used for functional testing of theprototypes on the BenchMark ULTRA using the U OptiView DAB IHC v5.2 CDxprocedure Procedure Step PTEN (SP218) CD34 (QBEnd/10) DeparaffinizationSelected Selected Cell Conditioning (CC1) 100° C., 56 minutes none Preantibody peroxidase Selected Selected inhibitor Antibody Incubation HandTitration Hand Titration Selected, Selected, 16 minutes, 37° C. 16minutes, 37° C. OptiView HQ Linker 8 minutes (default) 8 minutes(default) OptiView HRP Multimer 8 minutes (default) 8 minutes (default)Hematoxylin II 4 minutes 4 minutes Bluing Reagent 4 minutes 4 minutes

Referring to Table 2, unstained slides of normal human prostate orplacenta tissues were placed in BenchMark ULTRA instruments and stainedwith PTEN (SP218) and CD34 (QBEnd/10), respectively. The PVA filmprototypes were placed manually onto the slide during the “hand applyantibody titration” step, where the instrument allows the user to openthe slide drawer to manually apply antibody into the reaction bufferpuddle on top of the slide. Controls for each run were included, where anormal dispenser was used to dispense the antibody onto the slideinstead of the PVA prototype. Reaction buffer was also pipetted on topof the PVA film (approximately 400 μL) in order to compensate for bufferdisplaced by placement of the prototype, and to speed up PVAdissolution. Although this reaction buffer step was performed manually,it could be added to the staining procedure by changing the hand applysoftware macro to include a reaction buffer “adjust” step after closingthe slide drawer. After each staining run, the slides were dehydratedand coverslipped according to the VENTANA OptiView DAB Detection Kitpackage insert (1010323EN, Ventana Medical Systems, Tucson, Ariz.)).

B. Results for Antibody Embedded Wafer Composition

Referring to FIG. 9A, FIG. 9B, FIG. 9C, and FIG. 9D, the antibodyembedded PVA film wafer was made with the proper concentrations ofdiluent components by adding the antibody to the PVA solution directlyfrom a dispenser. In addition, formulating the PVA film promotedpreliminary optimization of film composition. FIG. 9A illustrates anormal prostate tissue stained with PTEN(SP218) antibody from 1% PVAantibody embedded film while FIG. 9B illustrates normal prostate tissuestained with PTEN(SP218) antibody dispenser as a control. The slides inFIGS. 9A and 9B were comparably stained. FIG. 9C is a 40× magnificationof the slide of FIG. 9A while FIG. 9D is a 40× magnification of theslide of FIG. 9B. Staining from each of the 1-4% PVA film wafers wereconsistent with the dispenser control; however, the 3% and 4% PVA filmsdid not fully dissolve during the staining run, and remnants were foundat the base of the slide after the staining run was complete.Dehydration of the antibody-PVA solution at 45° C. did not appear toaffect antibody stability or function. On the other hand, dehydration at60° C. led to antibody denaturation and loss of functional staining.Dehydration at lower temperatures, or even lyophilization, may improvestability and improve antibody function if some antibodies are found tobe sensitive to heat.

C. Results for Antibody Packet Composition

Initial staining from the antibody packet was inconsistent, and stainingwas improved when packets were made thinner and more reaction buffer wasadded to help dissolve the packet. However, staining intensities acrossthe tissues remained inconsistent with small non-staining areas. FIG.10A, FIG. 10B, FIG. 10C, and FIG. 10D illustrate examples of tissuesstained with the PVA packet compared to a dispenser control. FIG. 10A isan image of normal prostate tissue stained with PTEN(SP218) antibodyusing PVA packet filled with glycerin and antibody while FIG. 10B is animage of normal prostate tissue stained with PTEN(SP218) antibody usinga dispenser as a control (FIG. 10B). FIG. 10C illustrates 40× images ofthe packet while FIG. 10D illustrates 40× images of the control,revealing similar staining quality. Glycerin appeared to sink to bottomof the puddle as it was released from the dissolving PVA film during thehand apply step, which may have prevented the antibody from reaching thetissue and binding appropriately across the entire slide. Air bubbleswere also observed when the packet was dissolving, and could also beblocking antibody from reaching some areas. Glycerin was chosen as adiluent for testing as it is readily commercially available,non-hazardous, and would not dissolve the surrounding PVA packet likeaqueous antibody diluent. Glycerin is highly viscous liquid that doesnot readily dissolve in aqueous solutions, and has a higher density thanwater.

D. Results for Inkjet Printed Antibody Film Composition

FIG. 11A, FIG. 11B, FIG. 11C, and FIG. 11D show examples of tissuesstained with the inkjet-printed antibody film compared to a dispensercontrol. Specific staining intensities from the inkjet-printed antibodyfilm was consistent with the dispenser control; however, morebackground, or nonspecific, staining was present. Background stainingmay be attributed to different concentrations of diluent components thatcould be optimized with future studies.

FIG. 11A illustrates normal placenta tissue stained with CD34 (QBEnd/10)primary antibody printed onto PVA film while FIG. 11B illustrates normalplacenta tissue stained with CD34 (QBEnd/10) primary antibody deliveredwith a dispenser as a control. FIG. 11C is a 40× magnification of theslide of FIG. 11A while FIG. 11D is a 40× magnification of the slide ofFIG. 11B. These slides reveal similar stain intensity and slightlyhigher background staining with the PVA film. Specific stainingintensities from the inkjet-printed antibody film were consistent withthe dispenser control; however, more background, or nonspecific,staining was present. Background staining may be attributed to differentconcentrations of diluent components.

Example 7

Example 7 describes an example of a Staining Protocol for use with solidcompositions of the present invention. The solid compositions used inthe example below include a solid composition comprising a first(primary) antibody, a solid composition comprising a second antibody, acomposition comprising a third antibody, a composition comprising afirst detection reagent, a composition comprising a second detectionreagent, a composition comprising hematoxylin, and a compositioncomprising a bluing reagent. The present invention is not limited to themethods, compositions, reagents, systems, or specific proceduresdescribed herein.

Apply deparaffinization solution

Apply antigen retrieval solution

Apply primary antibody solid composition

Apply secondary antibody solid composition

Apply third antibody solid composition

Apply first detection reagent solid composition

Apply second detection reagent solid composition

Apply Hematoxylin solid composition

Apply Bluing Reagent solid composition

REFERENCES

The disclosures of the following articles and patent documents areincorporated in their entirety by reference herein:

-   Butler J. E. “Solid Supports in Enzyme-Linked Immunosorbent Assay    and Other Solid-Phase Immunoassays” Methods 22 (2000):4-23-   De Melo-Junior M. R., Alves L. C., dos Santos F. B., Beltrão E. I.    C., de Carvalho Jr L. B. “Polysiloxane-polyvinyl alcohol discs as    support for antibody immobilization: Ultra-structural and    physical-chemical characterization” React Funct Polym 68    (2008):315-320-   Kennedy S. P., inventor; The Procter & Gamble Company, assignee.    “Liquid Laundry Detergent in Water-Soluble Package”. U.S. Pat. No.    4,973,416. Nov. 27, 1990-   McWilliam, I., M. Chong Kwan, and D. Hall. “Inkjet Printing for the    Production of Protein Microarrays.” Methods in Molecular Biology 785    (2011): 345-61.-   Robert, MC., M. Frenette, and C. Zhou. “A Drug Delivery System for    Administration of Anti-TNF-a Antibody.” Transnational Vision Science    & Technology 5.2 (2016)-   Sonenstein G. G., inventor; Colgate-Palmolive Company, assignee.    “Water Soluble Films Of Polyvinyl Alcohol And Polyacrylic Acid And    Packages Comprising Same”. U.S. Pat. No. 4,692,494. Sep. 8, 1987-   Stephens C, Suriyavirun N, inventors; Ventana Medical Systems,    assignee. “Solid Single-dose Antibody and Reagent Delivery Method    for IHC/ISH Instruments” U.S. Provisional Patent Application No.    62/437,545 Filed December 2016.-   Wan T., Stylios G. K., Giannoudi M., Giannoudis P. V. “Investigating    a new drug delivery nano composite membrane system based on PVA/PCL    and PVA/HA(PEG) for the controlled release of biopharmaceuticals for    bone infections.” Injury, Int. J. Care Injured 46 S8 (2015):S39-S43

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

What is claimed is:
 1. An automated system comprising: (a) a basesupporting a sample; (b) a liquid dispenser containing a liquid, whereinthe liquid dispenser aliquots the liquid onto the sample; and (c) asolid reagent dispenser containing a solid composition, wherein thesolid composition comprises a water-soluble polymer and a reagentembedded in the water-soluble polymer, wherein the reagent is anantibody, an antibody fragment, or a protein, and wherein thewater-soluble polymer comprises polyvinyl alcohol (PVA), dextran,hydroxypropyl, cellulose, poly (acrylic acid sodium salt), poly(ethylene glycol), poly (methylacrylic acid sodium salt),poly(styrenesulfonic acid sodium salt), pullulan, or a combinationthereof, wherein the solid reagent dispenser deposits the solidcomposition on top of the sample; and wherein when the solid compositionis deposited on top of the sample and the solid composition comes incontact with the liquid, the solid composition dissolves onto thesample, thereby depositing the reagent onto the sample.
 2. The system ofclaim 1, wherein the sample comprises a tissue section.
 3. The system ofclaim 1, wherein the water-soluble polymer is a film.
 4. The system ofclaim 1, wherein the reagent is embedded in the water-soluble polymer byinfusing the reagent in the water-soluble polymer.
 5. The system ofclaim 1, wherein the reagent is embedded in the water-soluble polymer byprinting the reagent on the water-soluble polymer.
 6. The system ofclaim 1, wherein the reagent is embedded in the water-soluble polymer byencapsulating the reagent with the water-soluble polymer.
 7. The systemof claim 1, wherein the solid composition further comprises a secondreagent embedded in the water-soluble polymer, wherein the secondreagent is selected from a group consisting of: an antibody, an antibodyfragment, a protein, a nucleic acid, a chromogen, a stain, acounterstain, a lipid, a carbohydrate, an enzyme, a buffer, or acombination thereof.
 8. The automated system of claim 7, wherein thesecond reagent is a protein, and wherein the protein is an antibody oran antibody fragment.
 9. The system of claim 1, wherein the automatedsystem can further perform a histochemistry assay on the sample.
 10. Thesystem of claim 1, wherein the automated system can further perform insitu hybridization on the sample.
 11. The system of claim 1, wherein theautomated system can further perform automated DNA amplification or DNAsequencing.
 12. The automated system of claim 1, wherein the reagent isa protein, and wherein the protein is an antibody or an antibodyfragment.
 13. A solid composition, wherein the solid compositioncomprises: (a) a water-soluble polymer; and (b) a first reagent embeddedin the water-soluble polymer, and wherein the first reagent is anantibody, an antibody fragment, or a protein, and the water-solublepolymer comprises polyvinyl alcohol (PVA), dextran, hydroxypropylcellulose, poly (acrylic acid sodium salt), poly (ethylene glycol), poly(methylacrylic acid sodium salt), poly(styrenesulfonic acid sodiumsalt), pullulan, or a combination thereof.
 14. The solid composition ofclaim 13, wherein the water-soluble polymer is a film.
 15. The solidcomposition of claim 13, wherein the first reagent is embedded in thewater-soluble polymer by infusing the first reagent in the water-solublepolymer.
 16. The solid composition of claim 13, wherein the firstreagent is embedded in the water-soluble polymer by printing the firstreagent on the water-soluble polymer.
 17. The solid composition of claim13, wherein the first reagent is embedded in the water-soluble polymerby encapsulating the first reagent with the water-soluble polymer. 18.The solid composition of claim 13, wherein the first reagent isencapsulated by two or more layers of the water-soluble polymer.
 19. Thesolid composition of claim 13, wherein the solid composition furthercomprises salts from a buffer.
 20. The solid composition of claim 13,wherein the solid composition further comprises a buffering agent. 21.The solid composition of claim 13, wherein the solid composition furthercomprises a second reagent embedded in the water-soluble polymer,wherein the second reagent is selected from a group consisting of: anantibody, an antibody fragment, a protein, a nucleic acid, a chromogen,a stain, a counterstain, a lipid, a carbohydrate, an enzyme, a buffer,or a combination thereof.
 22. The solid composition of claim 21, whereinthe second reagent is a protein, wherein the protein is an antibody oran antibody fragment.
 23. The solid composition of claim 13, wherein thefirst reagent is a protein, wherein the protein is an antibody or anantibody fragment.